Chemical constituents of industrial hemp roots and their anti-inflammatory activities

Objective Although the chemical constituents of the aerial parts of Cannabis have been extensively studied, phytochemicals of Cannabis roots are not well characterized. Herein, we investigated the chemical constituents of industrial hemp (Cannabis sativa L.) roots and evaluated the anti-inflammatory activities of phytochemicals isolated from the hemp roots extract. Methods An ethyl acetate extract of hemp roots was subjected to a combination of chromatographic columns to isolate phytochemicals. The chemical structures of the isolates were elucidated based on spectroscopic analyses (by nuclear magnetic resonance and mass spectrometry). The anti-inflammatory effects of phytochemicals from hemp roots were evaluated in an anti-inflammasome assay using human monocyte THP-1 cells. Results Phytochemical investigation of hemp roots extract led to the identification of 32 structurally diverse compounds including six cannabinoids (1–6), three phytosterols (26–28), four triterpenoids (22–25), five lignans (17–21), and 10 hydroxyl contained compounds (7–16), three fatty acids (29–31), and an unsaturated chain hydrocarbon (32). Compounds 14–21, 23, 27, and 32 were identified from the Cannabis species for the first time. Cannabinoids (1–5) reduced the level of cytokine tumor necrosis-alpha (by 38.2, 58.4, 47.7, 52.2, and 56.1%, respectively) and 2 and 5 also decreased the interleukin-1β production (by 42.2 and 92.4%, respectively) in a cell-based inflammasome model. In addition, non-cannabinoids including 11, 13, 20, 25, 29, and 32 also showed selective inhibition of interleukin-1β production (by 23.7, 22.5, 25.6, 78.0, 24.1, 46.6, and 25.4%, respectively) in THP-1 cells. Conclusion The phytochemical constituent of a hemp roots extract was characterized and compounds from hemp roots exerted promising anti-inflammatory effects. Supplementary Information The online version contains supplementary material available at 10.1186/s42238-022-00168-3.


Introduction
The medicinal uses of Cannabis roots for a variety of maladies are supported by empirical practice and emerging scientific evidence. The ancient Chinese herbology Compendium of Materia Medica ("Bencao Gangmu" in Chinese or Great Pharmacopoeia) was one of the earliest books that documented the use of hemp (Cannabis sativa L.) roots as a folk medicine to soothe the pain. The traditional Chinese medicine system also uses hemp roots as a common practice to treat Reqi ("hot air" or heatiness)-related symptoms (e.g., mouth ulcers) by indigenous people from Southwest China regions. Modern scientific studies provide more rigorous evidence of the biological activities of cannabis roots. Several preclinical studies reported that cannabis root extracts exert various pharmacological effects including anti-inflammatory, estrogenic, liver protective, and anti-cancer activities (Elhendawy et al., 2019;Kornpointner et al. 2021;Lima, et al., 2021;Ryz et al., 2017). Notably, there are fewer reported studies on cannabis roots as compared to the plant's aerial parts including the inflorescences and leaves. This is possibly due to the lower density of glandular trichomes in cannabis roots, which leads to a lower level of cannabinoids biosynthesis (Stout et al., 2012). Moreover, the mechanisms of actions of enzymes that contribute to the biosynthesis of phytochemicals in the roots of cannabis are not fully elucidated (Sirikantaramas and Taura 2017). Nevertheless, several studies on hemp roots have reported the isolation and identification of several phytochemicals including cannabinoid-type of compounds, such as cannabidiol (CBD), tetrahydrocannabinol (THC), cannabichromene, cannabigerol, and cannabigerolic acid, as well as non-cannabinoid-type of compounds including flavonoids, triterpenes, polyphenols, and alkaloids (Stout et al., 2012). In addition, a study characterized the cannabinoids content (ranging between 0.001 and 0.004%) in three chemovars, which was lower than the other chemotypes of compounds such as sterols (0.06-0.09%) and triterpenoids (0.13-0.24%) (Jin et al. 2020). Notably, some unique compounds have been isolated from cannabis roots. For instance, a phenolic amide, namely, N-trans-coumaroyltyramine, was isolated from the roots of a Cannabis sp. grown in Mississippi, USA (Pollastro et al., 2018). In addition, several arylnapthalene-type of compounds including cannabisins B and G were only found in fruits and roots of cannabis (Sakakibara et al., 1995). However, to date, the phytochemical constituents of hemp roots and their biological activities are not been fully investigated. Our group has initiated a program to systematically investigate the biological activities of non-psychedelic cannabinoids Puopolo et al., 2022). During this course, we revealed that the anti-inflammatory activity of CBD is associated with its inhibitory effect on the activation of NLR-family-pyrin-domain-containing 3 (NLR3) inflammasome, which is a vital molecular target for the production of pro-inflammatory cytokine (i.e., interleukin 1 beta; IL-1β) . However, the phytochemical constituents of hemp roots from the region of Southwest China (i.e., Yunnan Province) and the inhibitory effects of cannabinoids from hemp roots are still not clear. Herein, we report the isolation and identification of phytochemicals of hemp roots as well as the biological evaluation of isolated phytochemicals from hemp roots using a cellbased inflammasome model.

Chemical reagents
The roots of industrial hemp were provided by Mawang Shenzhen Co. Ltd. (Shenzhen, China) and a voucher sample (No. MG20191230) is deposited in the School of Pharmaceutical Sciences of Sun Yat-Sen University. Lipopolysaccharides (LPS), nigericin, phorbol 12-myristate 13-acetate (PMA), and phosphate-buffered saline were purchased from Sigma-Aldridge Chemical Co (St. Louis, MO, USA).

Cell culture
Human monocyte THP-1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in the Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Gaithersburg, MD, USA).

Inflammasome inhibition assay
The anti-inflammasome effects of cannabinoids isolated from hemp root were evaluated by measuring the production of lipopolysaccharide (LPS)-and nigericininduced secretion of pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using a previously reported method ). Briefly, THP-1 cells were seeded at a density of 5 × 10 4 cells per well in a 48-well plate and differentiated with phorbol 12-myristate 13-acetate (PMA) (25 nM) for 48 h. Then, culture medium was removed and replaced with PMA-free medium for 24 h. Next, LPS (100 ng/ mL) was added and incubated for 4 h, followed by adding hemp root samples (compounds 1-5; 50 µM) and further incubated for 1 h. Then, nigericin (10 µM) was added and incubated with the cells for 4 h. The cell culture supernatant was collected to measure the levels of IL-1β and TNF-α using the ELISA kits (BioLegend, San Diego, CA, USA).
Apart from cannabinoid-type compounds (1-6), several non-cannabinoid compounds were identified from hemp roots. These phytochemicals included three phytosterols (26-28), four triterpenoids (22-25), and five lignans (17-21). Several compounds were isolated and identified from Cannabis sp. for the first time as follows. Compounds 20, 21, 23, and 27 were reported from the Cannabis species for the first time. Additionally, 10 hydroxyl-contained compounds (7-16) were isolated from the hemp roots. Among these, compounds 14-19 were identified from Cannabis for the first time. Moreover, three fatty acids (29-31) and an unsaturated hydrocarbon (32) were identified in Cannabis for the first time.

Anti-inflammatory effects of phytochemicals isolated from hemp roots
Our group has previously reported that CBD, the major non-psychedelic cannabinoid in cannabis, confers the anti-inflammatory effect via a specific mechanism, namely, inhibition of inflammasome activation . However, it is not clear whether other cannabinoids from the hemp roots are inflammasome inhibitors. Thus, an inflammasome inhibition assay was performed to evaluate the anti-inflammatory effects of cannabinoids isolated from hemp roots. The production of IL-1β and TNF-α in inflammasome inducers (LPS and nigericin) stimulated THP-1 cells were significantly increased as compared to the control group (6.1 vs. 1211.6 and 13.0 vs. 102.6 pg/mL, respectively), suggesting that inflammasome in THP-1 cells was activated by the stimulation with LPS and nigericin. Compounds 2 and 5 (50 µM) counteracted LPS-nigericin-induced production of IL-1β by 42.2% and 92.4%, respectively ( Fig. 2A). Cannabinoids (1-5) reduced the level of TNF-α by 38.2%, 58.4%, 47.7%, 52.2%, and 56.1%, respectively (Fig. 2B). To confirm whether the anti-inflammatory of these cannabinoids were selective inhibitors of the inflammasome, we used a small molecule with known a selective inflammasome inhibitor effect (Wu et al., 2020), namely, MCC950, as a positive control. MCC950 (10 µM) showed a specific inhibitory effect against the activation of the inflammasome by reducing the level of IL-1β (by 95.8%) without decreasing the level of TNF-α. Additionally, a small molecule with inhibitory effects on the production of TNF-α (DaSilva et al., 2019;Xu et al., 2018), namely, urolithin A, was included as a positive control (as a TNF-α inhibitor) Fig. 1 Chemical structures of compounds 1-32 including cannabinoids (1-6) and non-cannabinoids (7-32) isolated from hemp roots to validate the anti-inflammatory assay. Urolithin A reduced the level of TNF-α by 51.7% as compared to the model group. With these validated bioassays, we were able to evaluate the selective anti-inflammasome activity of the isolates. Although our previously reported study showed that CBD is a specific inflammasome inhibitor , cannabinoids isolated from hemp roots extract exerted general anti-inflammatory effects rather than specific inhibition of inflammasome activation. The inhibitory effects of cannabinoids from hemp roots on the secretion of pro-inflammatory cytokine (TNF-α) may be accounted for the overall anti-inflammatory activities of Cannabis roots extracts in previously reported studies (Lima, et al., 2021;Ryz et al., 2017). However, several factors that may influence the anti-inflammatory effects of tested cannabinoids should be examined. For instance, the cytotoxicity of these cannabinoids in THP-1 cells may be accounted for the reduction of the pro-inflammatory cytokines. Thus, we assessed the viability of THP-1 cells treated with cannabinoids. Compounds 1-4 showed no significant cytotoxic effect (viability > 95%) at the concentration used for the anti-inflammasome assay (50 µM) whilst cannabivarin (5) reduced the cell viability by 54.7%, which possibly contributed to its suppressive effect on the production of IL-1β and TNF-α (see cell viability data in the Supplementary Data Figure S1). Therefore, further mechanistic investigation on the anti-inflammatory effects of cannabinoids from hemp roots should be conducted at non-cytotoxic concentrations. Nevertheless, this is the first study on the specific anti-inflammasome activity of cannabinoids isolated from hemp roots.
In summary, a phytochemical investigation of a hemp roots extract led to the identification of 32 compounds including six cannabinoids (1-6) and 26 noncannabinoid compounds. Isolates 14-21, 23, 27, and 32 were reported from Cannabis for the first time. Cannabinoids from the hemp roots extract exerted antiinflammatory effects by reducing pro-inflammatory cytokines in an inflammasome model with THP-1 cells. Cannabinoids 2 and 5 reduced LPS-nigericininduced production of cytokines IL-1β (by 42.2% and 92.4%, respectively) and TNF-α (by 58.4% and 56.1%, Fig. 2 Anti-inflammatory effects of cannabinoids (1-5) isolated from hemp roots. Inflammasome activation in THP-cells was induced by the stimulation with LPS-nigericin. Effects of 1-5 (at 50 µM) on the production of pro-inflammatory cytokines including IL-1β (A) and TNF-α (B). Data are presented as means ± standard deviation from samples with three replicated experiments (n = 3). Statistical analysis was performed with a one-way analysis of variance with multiple comparisons and the significance was noted as: ***p < 0.001, ****p < 0.0001 vs. the model (LPS-nigericin stimulated) group and ####p < 0.0001 vs. the control group respectively). In addition, non-cannabinoids type compounds including 11,13,19,20,25,29, and 32 also showed promising anti-inflammatory effects. Findings from the current study expanded the understanding of the phytochemical constituents of cannabis roots and their anti-inflammatory effects, which may provide useful information for the utilization of cannabis roots as bioactive natural products.

Acknowledgements
Spectroscopic instruments used for the completion of the biological evaluations in this study were located in the RI-INBRE core facility supported by Grant # P20GM103430 from the National Institute of General Medical Sciences of the National Institutes of Health.
Authors' contributions HM and QG conceived and designed the overall project; SH conducted the phytochemical isolation and identification; HL conducted the biological evaluations; JX, HZ, and NPS supervised the study; SH and HL wrote the manuscript; HM and QG extensively edited the manuscript. The author(s) read and approved the final manuscript.

Authors' information
Not applicable.

Funding
The phytochemical investigation including the isolation and identification of compounds in the hemp root extract was supported by a research contract between Mawang Shenzhen Co. Ltd and Sun Yat-sen University (HT-99982019-0530).

Availability of data and materials
The data used for this study are available from the corresponding authors with a reasonable request.

Declarations
Ethics approval and consent to participate Not applicable. No human subjects involved in this study.

Consent for publication
Not applicable. No human subjects involved in this study.

Competing interests
J.X. received funding from a research contract between Mawang Shenzhen Co. Ltd and Sun Yat-sen University (HT-99982019-0530). Mawang Shenzhen provided the materials of hemp roots but did not involve in the design, investigation, and presentation of this study. Other authors declare no competing interests.